Figure 6.

A3G-3A co-purifies with viral nucleoprotein complexes. Virus stocks were made in HeLa cells by cotransfection of pNL4-3 Vif (-) plasmid DNA (3 μg each) with 2 μg each of pcDNA-A3A (panel A), pcDNA-A3G (panel B), or pcDNA-A3G-3A DNA (panel C). Virus containing supernatants were collected 24 h post-transfection, filtered to remove cellular debris, and concentrated by pelleting through 20% sucrose. Viral pellets were suspended in 1 ml of DMEM and 500 μl each of the virus preparation was loaded onto a 20%/60% sucrose step gradient previously overlaid with 100 μl of PBS (lanes 1–3) or Triton X-100 (lanes 4 to 6) as described in Materials and Methods. Three fractions of 1.1 ml each were collected from the top of the gradient as shown in the cartoon on the right. Fraction S1 (lanes 1 & 4) contains soluble proteins; fraction S2 (lanes 2 & 5) is a buffer fraction of 20% sucrose that separates soluble proteins from virus particles or viral cores; fraction S3 (lanes 3 & 6) includes the interphase of 20%:60% sucrose where viral particles and viral cores accumulate. Gradient fractions were subjected to immunoblot analysis using an A3G-specific antibody (A3A, A3G, or A3G-3A) followed by probing with an HIV-positive patient serum (CA). Nucleocapsid protein (NC) was identified by a goat anti-NC antibody and matrix protein (MA) was identified by a mouse monoclonal antibody to MA(P17).